Tags

Type your tag names separated by a space and hit enter

Effect of residue substitution via site-directed mutagenesis on activity and steroselectivity of transaminase BpTA from Bacillus pumilus W3 for sitafloxacin hydrate intermediate.
Int J Biol Macromol 2019; 137:732-740IJ

Abstract

Aminotransferases are widely employed as biocatalysts for the asymmetric synthesis of biologically active pharmaceuticals. Transaminase BpTA from Bacillus pumilus W3 can accept a broad spectrum of sterically demanding substrates, but it does not process the key five-membered ring intermediate of sitafloxacin. In the present study, we rationally constructed numerous single-point mutants and six multi-point mutants by combining the structural characteristics of transaminase and its substrates. Biochemical characteristics of wild-type and mutant enzymes were initially analyzed, and mutants I215M, I215F, and Y32L displayed increased catalytic efficiency, K155A, I215V and T252A completely lost enzyme activity. Residues K155 and T252 had a particularly strong influence on catalytic activity. Four multi-point mutants (L212M/I215M, Y32L/S190A/L212M/I215M, Y32L/Y159F/T252A and Y32W/Y159F/I215M/T252A) possess potential for industrial production of the key five-membered ring intermediate of sitafloxacin. Furthermore, mutants Y32L/Y159F/T252A and Y32W/Y159F/I215M/T252A can catalyze conversion of (R)-α-phenethylamine, albeit at an extremely low rate (<8%). In summary, mutants L212M/I215M and Y32L/S190A/L212M/I215M are more suitable for industrial production of the antibiotic, sitafloxacin, via an enzymatic approach.

Authors+Show Affiliations

Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China.Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China.Chem-Stone (Guangzhou) Co., Ltd, Scientific and Technological Enterprise Accelerator, 11 Kaiyuan Avenue, Guangzhou 510530, China.Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China.Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China.Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China.Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China. Electronic address: yjcai@jiangnan.edu.cn.Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China. Electronic address: 13771104596@163.com.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

31279886

Citation

Zhai, Lixin, et al. "Effect of Residue Substitution Via Site-directed Mutagenesis On Activity and Steroselectivity of Transaminase BpTA From Bacillus Pumilus W3 for Sitafloxacin Hydrate Intermediate." International Journal of Biological Macromolecules, vol. 137, 2019, pp. 732-740.
Zhai L, Yang S, Lai Y, et al. Effect of residue substitution via site-directed mutagenesis on activity and steroselectivity of transaminase BpTA from Bacillus pumilus W3 for sitafloxacin hydrate intermediate. Int J Biol Macromol. 2019;137:732-740.
Zhai, L., Yang, S., Lai, Y., Meng, D., Tian, Q., Guan, Z., ... Liao, X. (2019). Effect of residue substitution via site-directed mutagenesis on activity and steroselectivity of transaminase BpTA from Bacillus pumilus W3 for sitafloxacin hydrate intermediate. International Journal of Biological Macromolecules, 137, pp. 732-740. doi:10.1016/j.ijbiomac.2019.07.027.
Zhai L, et al. Effect of Residue Substitution Via Site-directed Mutagenesis On Activity and Steroselectivity of Transaminase BpTA From Bacillus Pumilus W3 for Sitafloxacin Hydrate Intermediate. Int J Biol Macromol. 2019 Jul 4;137:732-740. PubMed PMID: 31279886.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Effect of residue substitution via site-directed mutagenesis on activity and steroselectivity of transaminase BpTA from Bacillus pumilus W3 for sitafloxacin hydrate intermediate. AU - Zhai,Lixin, AU - Yang,Shaolan, AU - Lai,Yingjie, AU - Meng,Di, AU - Tian,Qiaopeng, AU - Guan,Zhengbing, AU - Cai,Yujie, AU - Liao,Xiangru, Y1 - 2019/07/04/ PY - 2019/02/19/received PY - 2019/05/24/revised PY - 2019/07/03/accepted PY - 2019/7/8/pubmed PY - 2019/7/8/medline PY - 2019/7/8/entrez KW - Bioinformatics KW - Enzyme kinetics KW - Protein engineering KW - Stereoselective synthesis KW - Transaminase SP - 732 EP - 740 JF - International journal of biological macromolecules JO - Int. J. Biol. Macromol. VL - 137 N2 - Aminotransferases are widely employed as biocatalysts for the asymmetric synthesis of biologically active pharmaceuticals. Transaminase BpTA from Bacillus pumilus W3 can accept a broad spectrum of sterically demanding substrates, but it does not process the key five-membered ring intermediate of sitafloxacin. In the present study, we rationally constructed numerous single-point mutants and six multi-point mutants by combining the structural characteristics of transaminase and its substrates. Biochemical characteristics of wild-type and mutant enzymes were initially analyzed, and mutants I215M, I215F, and Y32L displayed increased catalytic efficiency, K155A, I215V and T252A completely lost enzyme activity. Residues K155 and T252 had a particularly strong influence on catalytic activity. Four multi-point mutants (L212M/I215M, Y32L/S190A/L212M/I215M, Y32L/Y159F/T252A and Y32W/Y159F/I215M/T252A) possess potential for industrial production of the key five-membered ring intermediate of sitafloxacin. Furthermore, mutants Y32L/Y159F/T252A and Y32W/Y159F/I215M/T252A can catalyze conversion of (R)-α-phenethylamine, albeit at an extremely low rate (<8%). In summary, mutants L212M/I215M and Y32L/S190A/L212M/I215M are more suitable for industrial production of the antibiotic, sitafloxacin, via an enzymatic approach. SN - 1879-0003 UR - https://www.unboundmedicine.com/medline/citation/31279886/Effect_of_residue_substitution_via_site-directed_mutagenesis_on_activity_and_steroselectivity_of_transaminase_BpTA_from_Bacillus_pumilus_W3_for_sitafloxacin_hydrate_intermediate L2 - https://linkinghub.elsevier.com/retrieve/pii/S0141-8130(19)31263-2 DB - PRIME DP - Unbound Medicine ER -